Alcohol use disorders (AUDs) have a staggering socio-economic impact, yet current therapeutic strategies are largely inadequate, due, in part, to a lack of information regarding the molecular target(s) on which ethanol acts. The current proposal tests the hypothesis that P2X4 receptors (P2X4Rs) represent a novel target for the development of drugs to prevent and/or treat AUDs. This target has not yet been explored. We have assembled a multidisciplinary team of investigators that together will use state of the art genetic, molecular, chemical and behavioral techniques to systematically test for targets in the mesolimbic dopamine (DA) system that will be amenable for drug development. The proposed studies will translate laboratory findings into opportunities to discover and develop novel therapeutics for the prevention and treatment of AUDs. Aim 1 studies will test the hypothesis that P2X4Rs within the brain dopamine reward systems regulate ethanol intake. Study 1, we will evaluate the effects of constitutive knockout of the p2rx4 gene (P2X4KO) on alcohol intake (24 hr access and drinking in the dark [DID] tests). Study 2, using targeted delivery of lentiviral-mediated short hairpin RNA (shRNA), we will knockdown P2X4R expression in the nucleus accumbens (NAc) and/or ventral tegmental area (VTA) to gain insights into specific contributions of the mesolimbic DA system regarding short- term effects of P2X4R reduction/blockade. This work will also help clarify the role of P2X4Rs in causing or modulating the effects of ethanol without the potential developmental alterations that may accompany constitutive KOs. We will use a new, selective P2X4R antagonist, 5-BDBD and develop new related probes, as a third method to determine P2X4Rs role in intoxication. Successful pharmacological manipulation of P2X4Rs would provide new insights into the importance of P2X4Rs as a viable target for future therapies for AUDs. Study 3, using molecular biological, brain slice electrophysiology and fast scan cyclic voltammetry we will begin to investigate the role of P2X4Rs in the NAc and VTA of the mesolimbic DA system. This work will provide mechanistic insights regarding P2X4Rs in the mesolimbic DA system and how they interact to modulate ethanol intake and behavior in mice (Studies 1 and 2). Aim 2 studies will use ivermectin (IVM), a positive modulator of P2X4Rs, in combination with P2X4KO mice, to test the hypothesis that stimulation of P2X4Rs will decrease ethanol intake. In addition, we will design, synthesize and test new chemical entities with positive, negative or null activity on P2X4R that can serve as pharmacologic probes to interrogate the P2X4R system. Taken together, the work will further our long term goal of identifying neurochemical mechanisms and key brain regions that control alcohol intake. Moreover, this work will identify new targets and will begin to develo new compounds for the translational development of therapeutic agents to prevent and or treat AUDs.